In living systems, there are many ion-transporters such as electrogenic pump molecules and carrier-mediated ion-exchangers in cell membranes. Functions of these ion-transporters are to maintain the specific ionic concentrations in the cell as well as the ionic concentration gradients across the cell membrane. These ionic concentration gradients result in an electrical potential across the cell membrane. The ionic concentration gradients and the membrane potential constitute electrochemical potential across the cell membrane, which is critical to many cell functions.
This electrochemical potential is the source for the generation and propagation of the action potential for all of the excitable cells, such as nerve cells, skeletal muscle fibers, and cardiac cells. This electrochemical potential also provides energy to many other membrane active-transporters, such as the Na/H exchangers which influence pH value. The ionic concentration gradients also play a significant role in controlling the cell volume and homeostasis. Therefore, maintaining the ionic concentration gradients and the membrane potential is critical to living cells.
The Na/K pump or Na/K ATPase is one of the most prevalent house-keeping proteins and is found within the membrane of almost every cell. It famously extrudes three Na ions out of the cell via the exchange of two K ions and consumption of one ATP in each pumping cycle in order to maintain the ionic concentration gradients and the membrane potential. The energy requirements of the Na/K pumps can constitute 20-80% of the cell's resting metabolic rate depending on the extent of electrical activity of the tissue.
Because stable ionic concentration gradients and membrane potential differences maintained by the Na/K pumps are critical to cell functions and survivability, any functional reduction of the pump molecules will inevitably affect cell functions and may result in cell necrosis and death. There are many situations where the functions of Na/K pumps can be significantly affected. One category of diseases includes those in which there is a lack of ATP molecules. Because a large amount of ATP molecules are used by Na/K pumps, lack of ATP molecules may fail to fuel the pump molecules. Many diseases are in this category, such as cardiac diseases and brain ischemia. Due to lack of blood and oxygen, ATP molecules cannot be effectively generated in the mitochondria to fuel the Na/K pumps. As a result, K ions cannot be sufficiently pumped into the cell and Na ions are accumulated in the cell. The reduced ionic concentration gradients result in membrane potential depolarization. In cardiomyocytes, the ionic concentration gradient reduction and the membrane resting potential depolarization cause many symptoms, such as murmurs, irregular beating, and finally heart failure.
Another example of a lack of ATP molecules failing to fuel the Na/K pump is electrical injury. An intensive electrical shock may generate pore or pore-like structures in a cell membrane, known as electroporation, resulting in leakage of ions and many other biomolecules including ATP from the cell. Due to the lack of ATP molecules to fuel the pump molecules, the ionic concentration gradient cannot be quickly restored. Consequently, the electrically injured cells may be swollen, ruptured, and eventually die.
A second category of diseases in which the Na/K pumps are significantly affected are those in which the density of the Na/K pump molecules in the cell membrane is significantly reduced. In these diseases the residual pump molecules are not competent to maintain the ionic concentration gradients and membrane potential. A short list of these diseases includes myotonic dystrophy, diabetes, cystic fibrosis, central nervous system disorder, McArdle disease, and various aging diseases, such as Alzheimer's disease and Huntington's disease. For example, with regard to Huntington's Disease it has been found that the density of Na/K pump molecules in the brain neurons of those suffering from Huntington's disease may decline to as low as 30%.
Finally, in some diseases the natural mechanisms controlling the functions of Na/K pumps are affected to the point of malfunction of the pump molecules. For example, dysfunction of the pump molecules in the kidneys, peripheral nerves, blood vessels, and muscle fibers in diabetes patients are often due to both metabolic deficiencies and control mechanism defects. Similarly, one of the mechanisms underlying long-term hypertension is related to the level of endogenous ouabain-like compound (EOLC) in the body which is involved in the control of the Na/K pumps. In order to reinstate normal cell functions and reduce the symptoms of disease, effective and efficient control or restoration of the Na/K pump functions has become a central target for treatment.
Many pump molecules and ion-exchangers are sensitive to changes in membrane potential due to the transportation of ions across the cell membrane. The voltage-dependence of Na/K pump current has been widely studied from nerve cells (Rakowski et al, 1989), oocytes (Rakowski et al., 1991), cardiac muscles (Nakao and Gadsby, 1989; Gadsby and Nakao, 1989) and skeletal muscle fibers (Chen and Wu, 2002). The results have shown that Na/K pumps have a sigmoid shaped I-V curve, which exhibits a shallow slope, saturation behavior, and a negative slope when membrane potential is depolarized (Lauger and Apell, 1986; De Weer, et al., 1988; and Rakowski, et al., 1997). These results indicate that the sensitivity of the pump molecules to membrane potential is not particularly high and that the pump current has an upper limit. At normal physiological condition a membrane potential change, by natural mechanisms, can adjust the pump functions to maintain the concentration gradients and membrane potential. However, for many diseases and emergency situations, the natural mechanisms fail to exert a membrane potential change to adjust pump function and membrane potential depolarization results.
In the past few decades, significant efforts have been made to electrically activate the Na/K pumps. The pioneering work by Tsong and Teissie studied the Na and K pumping modes, separately, (Teissie and Tsong, 1980; Serpersu and Tsong, 1983) in red blood cells. They found that a weak oscillating electric field (20V/cm) at 1 MHz can activate the Na pumping mode but failed to facilitate the K pumping mode. Similarly at 1 KHz, the field can activate the K pumping mode but not the Na pumping mode.
Both sinusoidal electric field and random telegraph fluctuating (RTF) electric field have been used as the oscillating field (Xie et al, 1994). A resonance frequency theory was later developed (Markin et al, 1992) to interpret these results. Intrinsic oscillating frequencies may exist for the two pumping modes. When the applied field oscillating frequency resonates with the intrinsic frequency, electric energy will be transduced to the pump molecules to activate the corresponding pumping mode (Tsong and Astumian, 1986, 1987; Markin et. al., 1992; Robertson and Astumian, 1991).
Liu et al. (1990) and Xie et al. (1994) studied the electrical activation of the two transports separately (either Na pumping mode or K pumping mode, not both together). The activation of the whole pumping cycle was not shown. Based on their results of three-orders of difference in the optimal frequencies for the two transports, it is impossible to use one electric field to simultaneously activate both Na and K transports and therefore the whole pumping cycle. In contrast, the present invention activates the whole pumping cycle to accelerate the pumping rate.
Second, in their studies (Liu et al. 1990; Xie et al. 1994) an electric field with a discrete frequency was employed and it was found that there are two separate frequencies that are optimal for either the Na or the K pumping modes. In contrast, the present invention employs an electric field with a sweeping or modulating frequency which is significantly different from a discrete frequency.
In addition, (Liu et al. 1990; and Xie et al, 1994) a field-strength of 20 V/cm was used for red blood cells. Based on the 5 μm diameter of the red blood cells, the field-induced membrane potential is about 5 mV. This field-induced membrane potential is significantly smaller than the field-induced membrane potential used in the synchronization modulation method where 30 mV or higher is needed. The significant difference in the field-strength is because underlying mechanisms involved in the two techniques are fundamentally different.
The underlying mechanism of the resonance-frequency-window theory considers the existence of intrinsic frequency windows. When the frequency of an applied oscillating electric recognizes or matches the protein's intrinsic frequency, resonance occurs and the pump molecules can maximally absorb energy from the electric field. In contrast, the underlying mechanism of the present invention is that the pump's turnover rate is entrainable by a specially designed oscillating electric field. The concept of intrinsic frequencies is not included in the synchronization modulation method. When a well designed oscillating electric field with a frequency comparable to the pump's physiological turnover rate is applied to the cells, the Na-extrusion and K-influx is eventually trapped into positive and negative half-cycles, respectively. All the individual pumps operate at the same pumping pace as the oscillating electric field, i.e. the pumping rate is synchronized to the field frequency. By carefully maintaining the pump synchronization and gradually increasing (or decreasing) the field frequency, the pumping rate can be progressively re-synchronized to new frequencies, up to a defined value.
The outputs of the resonance-frequency-window theory also differ dramatically from those of the synchronization modulation method. In the resonance-frequency-window theory, when responding to an electric field with an optimal frequency, the corresponding pumping mode will be activated. The pumping mode cannot be deactivated or controlled to a defined value. In contrast, the synchronization modulation method allows not only the activation or deactivation of the pumping rate but also can control the pumping mode to a defined pumping rate. The resonance-frequency-window theory is only a simple phenomenon of energy absorption while the synchronization modulation method is a procedure of dynamic entrainment of the pump molecules.
A second theory has been proposed for the activation of pump molecules. This theory is known as the excitation-stimulation theory. Clausen, in an excellent review [Clausen, T., 2003, Na/K pump regulation and skeletal muscle contractility, Physiological Review, 83:1269-1324], has summarized the underlying mechanisms involved in excitation-stimulation-induced activation of the Na/K pumps. Activation of the Na/K pumps elicited by excitation-stimulation is most likely to reflect a rapid, but slowly reversible increase in the affinity of the Na/K pump for intracellular Na ions, possibly elicited by depolarization during the action potentials. This would allow for a more efficient clearance of Na from the cytoplasm and K from the extracellular phase. Another possible mechanism is due to the excitation-induced leakage of Na and K ions which increase the availability of ions to bind with the pump molecules [Clausen, T., and Nielsen, O. B., 1998, Rapid activation for the Na/K pump: mechanisms and functional significance, Bio. Skr. Dan. Vid. Selsk., 49:153-158].
Clausen et al showed that excitation-stimulation can activate the Na/K pump functions in skeletal muscles (1998 and 2003), where the stimulation opens the Na and K channels and therefore increases the ion-availability and their binding affinity to the pumps. This is actually the natural mechanism in adjusting the pumping functions to maintain the physiological ionic concentration and the membrane potential. No electrical energy is involved in the pump molecules. Excitation-stimulation is successful in the natural physiological situation but not in the extraordinary situation such as in disease or injury.
The electric field used in the excitation-stimulation method neither directly influences the pump molecules nor delivers electrical energy to the pumps. Because the channel currents are much larger than the pump currents, skeletal muscles still undergo a net loss of K ions and a net gain of Na ions (Sejersted et al., 2000, Kiernan et al, 2004, Moldovan and Krarup, 2006). The increased intra-neuronal Na ions may lead to reversal of the Na/Ca exchanger (Tatsumi and Katayama, 1995) and trigger the destruction of peripheral axons (Smith and Hall, 2001; Waxman, 2005). Therefore, in spite of its activation effect, the excitation-stimulation method has negative functional consequences. In contrast, the synchronization modulation method directly affects the pump molecules by precisely providing electric energy to the Na- and K-transports in the positive and negative half-cycles, respectively.
Other models to explain the underlying mechanisms have been proposed including a Brownian motion model (Astumian, 1997, Tsong, 2002, 2003) and a recent adiabatic pump model (Astumian, 2003). However, most of these studies are mainly hypothesis or theoretical analysis. As discussed above, the synchronization modulation method is significantly different from both the resonance-frequency-windows theory and the excitation-stimulation method in many aspects including the basic concept and underlying mechanisms, the approach, and the output.
To date, there is no practical technique available to non-invasively and effectively activate the pumping cycle or accelerate the pumping rate of the Na/K pumps or other carrier-mediated ion transporters. This may be due to the pump molecules not being particularly sensitive to the membrane potential as evidenced by a sigmoidal shaped I-V curve. It may also be due to the difficulty in electrically increasing the pump currents by simply depolarizing the membrane potential. Thus, there is needed in the art a mechanism by which to activate the entire pumping cycle (both Na and K transports) and accelerate or decelerate the pumping rates of the Na/K pumps or other carrier-mediated ion transporters.
The present invention discloses a method of controlling the entire operating (pumping) cycle of a plurality of carrier-mediated ion transporters. Controlling the ion transporters is accomplished by synchronizing the turnover rate of the individual carrier-mediated ion transporters through the application of a specially designed oscillating electric field at a frequency initially comparable to the natural turnover rate of the ion transporter. After the turnover rates of the individual ion transporters are synchronized, the turnover rates are modulated by gradually adjusting the synchronization frequency in order to control their running cycle. The synchronization modulation method of the present invention can effectively control the entire pumping cycle of a plurality of ion-transporters.